HPLC

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Presented by:Mukesh Kumar Prajapati 2K11/PTE/04

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What is chromatography
Chromatography (from Greek chroma "color" and graphein "to write") is the collective term for a set of laboratory technique for the separation of mixtures. The mixture is dissolved in a fluid called the "mobile phase", which carries it through a structure holding another material called the "stationary phase".

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. various constituents of the mixture travel at different speeds, causing them to The

separate. The separation is based on differential partitioning between the mobile and stationary phases

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Kinds of chromatography
Based on mobile phase 1. 2. Liquid Column Chromatography Gas Liquid Chromatography

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What is Liquid Chromatography?

Liquid chromatography is a separation technique that involves the placement (injection) of a small volume of liquid sample into a tube packed with porous particles (stationary phase) Where individual components of the sample are transported along the packed tube (column) by a liquid moved by gravity.

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The components of the sample are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles.

The separated components are collected at the exit of this column and identified by an external measurement technique, such as a spectrophotometer that measures the intensity of the color, or by another device that can measure their amount.

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Principles of Liquid Chromatography

Load sampl e Add solvent

Column containing Click to edit Master subtitle style stationary time Collect components phase

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Four Basic Liquid Chromatography

Basic liquid chromatography modes are named according to the mechanism involved in these liquid is the mobile phase:1. Liquid/Solid Chromatography (adsorption chromatography) Click to edit Master subtitle style A. Normal Phase LSC B. Reverse Phase LSC 2. Liquid/Liquid Chromatography (partition chromatography) A. Normal Phase LLC 10/28/12

What is HPLC?
H P L C

High Performance Liquid Chromatography

It is also called as High Pressure liquid chromatography Click to edit Master subtitle style

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HPLC is a separation technique that involves: -the injection of a small volume of liquid sample -into a tube packed with tiny particles (3 to 5 micron (m) in diameter called the stationary phase -where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump. Click to edit Master subtitle style

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These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. These separated components are detected at the exit of this tube (column) by a flow-through device (detector) that measures their amount. An output from this detector is called a liquid chromatogram.

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What Does a Liquid Chromatogram Look Like?

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What does a high pressure LC look like

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.
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.
1. Pump: The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatograph at a specific flow rate, expressed in milliliters per min (ml/min). Normal flow rates in HPLC are in the 1-to 2-mL/min range. Typical pumps pressures range of 6000-9000 psi (400-to 600-bar). During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient). . Click to edit Master subtitle style

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Gradient vs. Isocratic Conditions Isocratic

mobile phase solvent composition remains constant with time Best for simple separations Often used in quality control applications that support and are in close proximity to a manufacturing process Gradient mobile phase solvent (B) composition increases with time Best for the analysis of complex samples Often used in method development for unknown mixtures Linear gradients are most popular (for example, the gradient shown at Click to edit Master subtitle style right)

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. injector serves to introduce the liquid sample into the flow stream of the The
2. Injector: mobile phase. Typical sample volumes are 5-to 20-microliters. The injector must also be able to withstand the high pressures of the liquid system. An auto sampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical

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. Considered the heart of the chromatograph the columns stationary phase

3. Column: separates the sample components of interest using various physical and chemical parameters. The small particles inside the column are what cause the high back pressure at normal flow rates. The pump must push hard to move the mobile phase through the column and this resistance causes a high pressure within the chromatograph.

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4. Detector: (elute) from the column.

The detector can see (detect) the individual molecules that come out A detector serves to measure the amount of those molecules so that the chemist can quantitatively analyze the sample components. The detector provides an output to a recorder or computer that results in the liquid chromatogram(i.e., the graph of the detector response).

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2020

5. Computer:

. called the data system, the computer not only controls all the Frequently
modules of the HPLC instrument but it takes the signal from the detector and uses it to determine the time of elution (retention time) of the sample components (qualitative analysis) and the amount of sample (quantitative analysis).

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Sample Injection
how is a sample actually put into an LC system HPLC Manual Injector: 1.User manually loads sample into the injector using a syringe and then turns the handle to inject sample into the flowing mobile phase which transports the sample into the beginning (head) of the column, which is at high pressure Click to edit Master subtitle style

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Contd.
Manual injector

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Auto sampler 1.measures the appropriate sample volume, injects the sample then flushes the injector to be ready for the next sample, etc., until all sample vials are processed for unattended automatic operation .

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HPLC Columns Types

Analytical (internal diameter 1.0 -4.6-mm; lengths 15 250 mm] Preparative(i.d. > 4.6 mm; lengths 50 250 mm) Capillary(i.d. 0.1 -1.0 mm; various lengths) Nano (i.d. < 0.1 mm, or sometimes stated as < 100 m) Materials of construction for the tubing Stainless steel (the most popular; gives high pressure capabilities) Click to edit Master subtitle style Glass (mostly for bimolecular) PEEK polymer (biocompatible and chemically inert to most solvents)

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Cont.

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HPLC Columns Packing Materials Columns are packed with small diameter porous particles.

The most popular sizes are: 5-m, 3.5-m and 1.8-m Columns are packed using high-pressure to ensure that they are stable during use These porous particles in the column usually have a chemically bonded phase on their surface which interacts with the sample components to separate them from one another for example, C18 is a popular bonded phase

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The process of retention of the sample components (often called analyte) is determined by the choice of column packing and the selection of the mobile phase to push the analyte through the packed column.

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Separation Modes of HPLC

There are four major separation modes that are used to separate most compounds: 1.Reversed-phasec chromatography 2.Normal-phase and adsorption chromatography 3.Ion exchange chromatography 4.Size exclusion chromatography Click to edit Master subtitle style

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Reversed-Phase Chromatography is polar (RPC) The column packing is non-polar and the mobile phase

over 90% of chromatographers use this mode The technique can be used for non-polar, polar, ionizable and ionic molecules For samples containing a wide range of compounds, gradient elution is often used One begins with a predominantly water-based mobile phase and then adds organic solvent as a function of time. The organic solvent increases the solvent strength and elutes compounds that are very strongly retained on the RPC packing

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Normal Phase or Adsorption Chromatography

In this mode, the column packing is polar (e.g. silica gel, cyanopropyl-bonded, amino-bonded, etc.) and the mobile phase is non-polar (e.g. hexane, iso-octane, methylene chloride, ethyl acetate) Normal phase separations are performed less than 10% of the time. The technique is useful for: water-sensitive compounds geometric isomers cis-trans isomers class separations and chiral Click to edit Master subtitle style compounds

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Ion Exchange Chromatography In ion exchange, the column packing contains ionic groups(e.g. sulfonic,

tetraalkylammonium) and the mobile phase is an aqueous buffer (e.g. phosphate, format, etc.). Ion exchange is used by about 20%of the liquid chromatographers The technique is well suited for: the separation of inorganic and organic anions and cation in aqueous solution. Ionic dyes, amino acids, and proteins can be separated by ion exchange because such compounds are salt in brine water,

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Size Exclusion Chromatography (SEC) In SEC, there is no interaction between the sample compounds and the
column packing material. Instead, molecules diffuse into pores of a porous medium. Depending on their size relative to the pore size, molecules are separated. Molecules larger than the pore opening do not diffuse into the particles, while molecules smaller than the pore opening enter the particle and are separated. Large molecules elute first. Smaller molecules elute later

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The SEC technique is used by 10-15% of chromatographers, mainly for polymer characterization and for proteins. There are two modes:

non-aqueous SEC-Gel Permeation Chromatography (GPC) aqueous SEC-Gel Filtration Chromatography (GFC)]

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Detection in HPLC
There are many detection principles used to detect the compounds eluting from an HPLC column. The most common are: Spectroscopic Detection Refractive Index Detection Click Detection Fluorescence to edit Master subtitle style

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Spectroscopic Detection Ultraviolet (UV) Absorption

An ultraviolet light beam is directed through a flow cell and a sensor measures the light passing through the cell. If a compound elutes from the column that absorbs this light energy, it will change the amount of light energy falling on the sensor. The resulting change in this electrical signal is amplified and directed to a recorder or data system. Click to edit Master subtitle style A UV spectrum is sometimes also obtained which may aid in the identification of a compound or series of compounds.

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. MS detector senses a compound eluting from the HPLC column first by An

Mass Spectroscopy (MS) ionizing it then by measuring its mass and/or fragmenting the molecule into smaller pieces that are unique to the compound. The MS detector can sometimes identify the compound directly since its mass spectrum is like a fingerprint and is quite unique to that compound

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Refractive Index (RI) Detection provides a way to detect The ability of a compound or solvent to deflect light
it. The RI is a measure of molecules ability to deflect light in a flowing mobile phase in a flow cell relative to a static mobile phase contained in a reference flow cell. The amount of deflection is proportional to concentration. The RI detector is considered to be a universal detector but it is not very Click to edit Master subtitle style sensitive.

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Fluorescence Detection
Compared to UV-Vis detectors fluorescence detectors offer a higher sensitivity and selectivity that allows to quantify and identify compounds and impurities in complex matrices at extremely low concentration levels(trace level analysis). Fluorescence detectors sense only those substances that fluoresce Click to edit Master subtitle style

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What is HPLC used for?

Separation and analysis of non-volatile or thermally-unstable compounds HPLC is optimum for the separation of chemical and biological compounds that are non-volatile Typical non-volatile compounds are:

Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol) Salts like sodium chloride and potassium phosphate Proteins likeClick white or blood subtitle style egg to edit Master protein Organic chemicals like polymers (e.g. polystyrene, polyethylene) Heavy hydrocarbons like asphalt or motor oil Many natural products such as ginseng, herbal medicines, plant extracts Thermally unstable compounds such as trinitrotoluene (TNT), enzymes 10/28/12

Qualitative analysis HPLC

The identification(ID) of individual compounds in the sample; the most common parameter for compound ID is its retention time(the time it takes for that specific compound to elute from the column after injection); depending on the detector used, compound ID is also based on the chemical structure, molecular weight or some other molecular parameter. Click to edit Master subtitle style

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Contd..

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Quantitative Analysis
how much is there?; There are two main ways to interpret a chromatogram1.determination of the peak height of a chromatographic peak as measured from the baseline; 2.determination of the peak area (see figure below); In order to make a quantitative assessment of the compound, a sample with a known amount of the compound of interest is injected and its peak height or peak area isClick to editIn manysubtitle there is a linear relationship between measured. Master cases, style the height or area and the amount of sample.

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Preparation of Pure Compound(s)

-

By collecting the chromatographic peaks at the exit of the detector, and concentrating by the compound

(analyte)

removing/evaporating

the solvent, - a pure substance can be

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prepared

for

later

use

(e.g.

organic synthesis, clinical studies,

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Trace analysis
A trace compound is a compound that is of interest to the analyst but its concentration is very low, usually less than 1% by weight, often parts per million (ppm) or lower; the determination of trace compounds is very important in pharmaceutical, biological, toxicology, and environmental studies since even a trace substance can be harmful or poisonous; in a chromatogram trace substances can be difficult to separate or detect; Click to edit Master subtitle style high resolutions separations and very sensitive detectors are required.

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Contd.

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ApplicationProteins and polypeptides are subject to various types of molecular transformations that affect The biological activity and integrity The RP-HPLC column must allow the scientist to see these degradation products and. Baseline resolution of six hydrophobic proteins and resolves impurities in Click to edit impurities lysozyme and myoglobinMaster subtitle style

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Columns:- Discovery BIO Wide Pore C5, 15cm x 4.6mm, 5m (Cat. No. 568422-

U) Mobile Phase: 75:25, (0.1% TFA in water):(0.1% TFA in CH3CN); Flow Rate: 1.0mL/min Temp: ambient Detection: 220nm Injection: 12L in 0.1%TFA Gradient: 0-100%B in 25 min Click to edit Master subtitle style

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1. RNase

(13.7kDa, 1mg/mL) (12.4kDa, 1mg/mL) (14.3kDa, 1mg/mL) (67.0kDa, 2.5mg/mL) (18.8kDa, 1mg/mL) (45.3kDa, 3.5mg/mL)

2. Cytochrome c 3. Lysozyme 4. BSA 5. Myoglobin 6. Ovalbumin

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Thank you.

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